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Selection of porcine oocytes in vitro through brilliant cresyl blue staining in distinct incubation media

Published online by Cambridge University Press:  13 December 2016

Elisa C.S Santos ,
Jorgea Pradieé ,
Elisângela M. Madeira ,
Miriane M. Pereira ,
Bruna Mion ,
Rafael G. Mondadori ,
Arnaldo D. Vieira ,
Ligia M.C. Pegoraro  and
Thomaz Lucia Jr
Elisa C.S Santos
Affiliation:
ReproPel, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil. Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil.
Jorgea Pradieé
Affiliation:
ReproPel, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil. Faculdade de Veterinária, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil.
Elisângela M. Madeira
Affiliation:
ReproPel, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil. Faculdade de Veterinária, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil.
Miriane M. Pereira
Affiliation:
Embrapa Clima Temperado, 96010–900, Pelotas, RS, Brasil.
Bruna Mion
Affiliation:
ReproPel, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil. Faculdade de Veterinária, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil.
Rafael G. Mondadori
Affiliation:
ReproPel, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil. Instituto de Biologia, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil
Arnaldo D. Vieira
Affiliation:
ReproPel, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil. Faculdade de Veterinária, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil.
Ligia M.C. Pegoraro
Affiliation:
Embrapa Clima Temperado, 96010–900, Pelotas, RS, Brasil.
Thomaz Lucia Jr*
Affiliation:
ReproPel, Faculdade de Veterinária, Universidade Federal de Pelotas, 96010–900-Pelotas-RS, Brazil. Faculdade de Veterinária, Universidade Federal de Pelotas, 96010–900, Pelotas, RS, Brazil.
*
All correspondence to: Thomaz Lucia Jr. ReproPel, Faculdade de Veterinária, Universidade Federal de Pelotas, 96010–900-Pelotas-RS, Brazil. E-mail: thomaz@pq.cnpq.br
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Summary

Staining with brilliant cresyl blue (BCB) may be used for oocyte selection, but BCB staining itself and the most commonly used selection medium (DMPBS) may compromise the development of porcine oocytes in vitro. This study evaluated DNA fragmentation, nuclear maturation, the area of migration of cortical granules (CG) and embryo development for stained (BCB+) and unstained (BCB−) oocytes incubated in DMPBS and in a modified medium (ReproPel) tested for the first time. Unexposed (UN), BCB+ and BCB− oocytes were incubated composing six groups: DMPBS/UN; DMPBS/BCB+; DMPBS/BCB−; ReproPel/UN; ReproPel/BCB+; and ReproPel/BCB−. There were more BCB+ oocytes in ReproPel than in DMPBS (P < 0.05). The DNA fragmentation was evaluated for oocytes in DMPBS/BCB+, DMPBS/BCB−, ReproPel/BCB+, ReproPel/BCB− and in porcine follicular fluid (control). The frequency of oocytes with no DNA fragmentation was greatest (64.6%) in DMPBS/BCB+ and lowest in ReproPel/BCB+ and ReproPel/BCB− (26.8 and 34.1%, respectively) (P < 0.05). Nuclear maturation rates were greater (P < 0.05) for DMPBS/BCB+ (63.1%), ReproPel/UN (55.1%) and ReproPel/BCB+ (50.2%) than for DMPBS/UN (40.8%) and ReproPel/BCB− (35.5%). The area of CG was greater (P < 0.05) for ReproPel/BCB− (80.7%) and DMPBS/UN (77.6%) than for ReproPel/UN (34.7%). Cleavage rates for DMPBS/BCB+ and ReproPel/BCB+ were greater than for DMPBS/UN (P < 0.05). Blastocyst development rates were greatest (P < 0.05) for ReproPel/UN and ReproPel/BCB+. In both media, BCB staining was apparently unable to select competent oocytes, which likely occurred due to toxicity. Despite the similar nuclear maturation and area of CG compared with DMPBS, oocytes selected in ReproPel presented impaired DNA integrity.

Keywords

DNA fragmentationMaturationMediaPorcine oocytesToxicity
Type
Research Article
Information
Zygote , Volume 25 , Issue 1 , February 2017 , pp. 49 - 55
Copyright
Copyright © Cambridge University Press 2016 

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References

Akaki, Y., Yoshioka, K., Noguchi, M., Hoshi, H. & Funahashi, H. (2009). Successful piglet production in a chemically defined system for in-vitro production of porcine embryos: dibutyryl cyclic AMP and epidermal growth factor-family peptides support in-vitro maturation of oocytes in the absence of gonadotropins. J. Reprod. Dev. 55, 446–53.Google Scholar
Berthelot-Ricou, A., Perrin, J., Giorgio, C.D., De Meo, M., Botta, A. & Courbiere, B. (2011). Comet assay on mouse oocytes: an improved technique to evaluate genotoxic risk on female germ cells. Fert. Ster. 95, 1452–57.Google Scholar
Cheng, W.M., Sun, X.L., An, L., Zhu, S.E., Li, X.H., Li, Y. & Tian, J.H. (2007). Effect of different parthenogenetic activation methods on the developmental competence of in vitro matured porcine oocytes. Anim. Biotechnol. 18, 131–41.Google Scholar
Egerszegi, I., Alm, H., Rátky, J., Heleil, B., Brüssow, K.P. & Torner, H. (2010). Meiotic progression, mitochondrial features and fertilization characteristics of porcine oocytes with different G6PDH activities. Reprod. Fert. Dev. 22, 830–38.Google Scholar
Funahashi, H., Koike, T. & Sakai, R. (2008). Effect of glucose and pyruvate on nuclear and cytoplasmic maturation of porcine oocytes in a chemically defined medium. Theriogenology 70, 1041–47.Google Scholar
Funahashi, H., Cantley, T.C., Stumpf, T.T., Terlouw, S.L. & Day, B.N. (1994). Use of low-salt culture medium for in vitro maturation of porcine oocytes is associated with elevated oocyte glutathione levels and enhanced male pronuclear formation after in vitro fertilization. Biol. Reprod. 51, 633–39.Google Scholar
Holm, P., Booth, P.J., Schmidt, M.H., Greve, T. & Callesen, H. (1999). High bovine blastocyst development in a static in vitro production system using Sofaa medium supplemented with sodium citrate and myo-inositol. Theriogenology 52, 683–700.Google Scholar
Hong, J. & Lee, E. (2007). Intrafollicular amino acid concentration and the effect of amino acids in a defined maturation medium on porcine oocyte maturation, fertilization, and preimplantation development. Theriogenology 68, 728–35.Google Scholar
Kempisty, B., Jackowska, M., Piotrowska, H., Antosik, P., Wozna, M., Bukowska, D., Brüssow, K.P. & Jaskowski, J.M. (2011). Zona pellucida glycoprotein 3 (pZP3) and integrin β2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test. Theriogenology 75, 1525–35.Google Scholar
Kim, J., You, J., Hyun, S.H., Lee, G., Lim, J. & Lee, E. (2010). Developmental competence of morphologically poor oocytes in relation to follicular size and oocyte diameter in the pig. Mol. Reprod. Dev. 77, 330–39.Google Scholar
Kitagawa, Y., Suzuki, K., Yoneda, A. & Watanabe, T. (2004). Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation on in porcine embryos. Theriogenology 62, 1186–97.Google Scholar
Naito, K. & Toyoda, Y. (1991). Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes. J. Reprod. Fert. 93, 467–73.Google Scholar
Pawlak, P., Warzych, E., Chabowska, A. & Lechniak, D. (2014). Differences in cytoplasmic maturation between BCB+ and control porcine oocytes do not justify application of the BCB test for a standard IVM protocol. J. Reprod. Dev. 60, 28–36.Google Scholar
Pawlak, P., Pers-Kamczyc, E., Renska, N., Kubickova, S. & Lechniak, D. (2011). Disturbances of nuclear maturation in BCB positive oocytes collected from peri-pubertal gilts. Theriogenology 75, 832–40.Google Scholar
Roca, J., Martinez, E., Vazquez, J.M. & Lucas, X. (1998). Selection of immature pig oocytes for homologous in vitro penetration assays with the brilliant cresyl blue test. Reprod. Fert. Dev. 10, 479–85.Google Scholar
Romar, R., Coya, P., Gadea, J. & Rath, D., (2005). Effect of oviductal and cumulus cells on zona pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal spermatozoa. Anim. Reprod. Sci. 85, 287–300.Google Scholar
Santos, E.C.S., Sato, D., Lucia, T. Jr. & Iwata, H. (2015). Brilliant cresyl blue staining negatively affects mitochondrial functions in porcine oocytes. Zygote 23, 352–59.Google Scholar
Santos, D.B., Schiar, V.P.P., Ribeiro, M.C.P., Schwab, R.S., Meinerz, D.F., Allebrandt, J., Rocha, J.B.T., Nogueira, C.W., Aschner, M. & Barbosa, N.B.V. (2009). Genotoxicity of organoselenium compounds in human leukocytes in vitro . Mutation Res. 676, 21–6.Google Scholar
Schoevers, E.J., Colenbrander, B. & Roelen, B.A.J. (2007). Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes. Theriogenology 67, 1108–22.Google Scholar
Statistix®. 2013; Statistix 10 Analytical Software. Tallahassee, FL USA.Google Scholar
Takahashi, M., Saka, N., Takahashi, H., Kanai, Y., Schultz, R.M. & Okano, A. (1999). Assessment of DNA damage in individual hamster embryos by comet assay. Mol. Reprod. 54, 1–7.Google Scholar
Tatemoto, H., Sakurai, N. & Muto, N. (2000). Protection of porcine oocytes against apoptotic cell death caused by oxidative stress during in vitro maturation: role of cumulus cells. Biol. Reprod. 63, 805–10.Google Scholar
Vajta, G., Holm, P., Greve, T. & Callesen, H. (1997). The submarine incubation system, a new tool for in vitro embryo culture: a technique report. Theriogenology 48, 1379–85.Google Scholar
Wang, W.H., Abeydeera, L.R., Cantley, T.C. & Day, B.N. (1997). Effects of oocyte maturation media on development of pig embryos produced by in vitro fertilization. J. Reprod. Fert. 111, 101–8.Google Scholar
Wongsrikeao, P., Otoi, T., Yamasaki, H., Agung, B., Taniguchi, M., Naoi, H., Shimizu, R. & Nagai, T. (2006a). Effects of single and double exposure to brilliant cresyl blue on the selection of porcine oocytes for in vitro production of embryos. Theriogenology 66, 366–72.Google Scholar
Wongsrikeao, P., Otoi, T., Taniguchi, M., Karja, N.W.K., Agung, B., Nii, M. & Nagai, T. (2006b). Effects of hexoses on in vitro oocyte maturation and embryo development in pigs. Theriogenology 65, 332–43.Google Scholar
Yoon, K.W., Shin, T.Y., Park, J.I., Roh, S., Lim, J.M., Lee, B.C., Hwang, W.S. & Lee, E.S. (2000). Development of porcine oocytes from preovulatory follicles of different sizes after maturation in media supplemented with follicular fluids. Reprod. Fertil. Dev. 12, 133–39.Google Scholar
Yoshida, M., Mizoguchi, Y., Ishigaki, K., Kojima, T. & Nagai, T. (1993). Birth of piglet derived from in vitro fertilization of pig oocytes matured in vitro. Theriogenology 39, 1303–11.Google Scholar
Yoshioka, K., Suzuki, C. & Onishi, A. (2008). Defined system for in vitro production of porcine embryos using a single basic medium. J. Reprod. Dev. 54, 208–13.Google Scholar